Design and generation of plasmids for the overexpression of microRNA* species.

<p>(A) Design of the stem-loop miRNA precursor. The mature sequences of miRNA* species were incorporated into the 3′ arm of the precursor, whereas the complementary sequences of the miRNA* species were incorporated into the 5′ arm. Note that the complementary sequences could either be completely complementary to the miRNA* species or could be modified by mutating several bases in its seed region, which were not complementary to the miRNA* species. (B) The designed hsa-miR-146b-3p stem-loop precursor. The red characters in the 3′ arm correspond with the sequences of hsa-miR-146b-3p. The 5′ arm sequences are the modified complementary sequences of hsa-miR-146b-3p, and the sequences above are hsa-miR-146b-5p, with blue characters denoting seed sequences and solid lines between them indicating the similarity of the two sequences. The dotted lines show the modified sites in the 5′ arm sequences and their corresponding nucleotides in hsa-miR-146b-3p sequences. (C) The uppermost oligo is the DNA insert of hsa-miR-146b-3p precursor obtained by annealing two single stranded DNA oligos with <i>Bam</i>H1 and <i>Eco</i>R1 sticky ends. The DNA insert was incorporated into plvx-shRNA2 between the recognition sites for the restriction enzymes <i>Bam</i>H1 and <i>Eco</i>R1. The representation of the vector was modified from an illustration in the instructions provided with the product.</p

A Novel Vector-Based Method for Exclusive Overexpression of Star-Form MicroRNAs

<div><p>The roles of microRNAs (miRNAs) as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*), which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA) overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like natural miRNAs, the overexpressed hsa-miR-146b-3p inhibited target gene expression by 3′UTR seed pairing. Our demonstration that this method could overexpress two other miRNAs suggests that the approach should be broadly applicable. Our novel strategy opens the way for exclusively stable overexpression of miRNA* species and analyzing their unique functions both <em>in vitro</em> and <em>in vivo</em>.</p> </div

Phosphorylation of STAT5 was observed in patients at the beginning and after treatment.

<p>The expression of phosphorylated STAT5 in B cells was notably reduced in patients who had achieved significant clinical improvement after treatment. SLE-04, SLE-05, SLE-10, and SLE-12 represent four different patients.</p

Demographics of SLE and RA patients and healthy donors.

<p>Except where otherwise indicated, the values expressed are means ± standard errors of the means (ranges). There were no significant differences between the patients with SLE, the patients with RA, and the healthy donors in terms of their age and sex. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; SLEDAI-2K, SLE Disease Activity Index 2000; HDs, healthy donors.</p

pSTATs in SLE patients after cytokine stimulation.

<p>The MFI for the phosphorylation of STAT1 (5A), STAT3 (5B), and STAT5 (5C) in the T cells of SLE patients decreased significantly after IFNα stimulation for 15 min. The MFI for the phosphorylation of STAT1 (5D), STAT3 (5E), and STAT5 (5F) decreased in the B cells of SLE patients after IFNα stimulation for 15 min. The MFI for the phosphorylation of STAT1 (5G) and STAT3 (5H) in the monocytes of SLE patients decreased after IFNα stimulation for 15 min. The MFI for pSTAT3 (5I) decreased significantly in the T cells of SLE patients after IL6 stimulation for 15 min. MFI, mean fluorescence index; SLE, systemic lupus erythematosus; HD, healthy donors; IFNα, interferon α.</p

Successful overexpression of hsa-miR-146b-3p by plvx-hs-146b-3p with no detectable increase of hsa-miR-146b-5p.

<p>HeLa cells were transfected with the indicated plasmids (400 ng per well) and RNA was collected and extracted 24 h later. The expression of hsa-miR-146b-3p (A) and hsa-miR-146b-5p (B) was detected using qRT–PCR. The same experiments were done in Hep G2 cells (C) for hsa-miR-146b-3p; (D) for hsa-miR-146b-5p and HEK 293T cells (E) for hsa-miR-146b-3p; (F) for hsa-miR-146b-5p. Each graph shows the mean of three independent experiments that measured the relative expression levels (2^−deltaCT) of the two miRNAs to the reference gene RNU48. Error bars represent SEMs. * means p value ≤0.05; ** means p value ≤0.01; *** means p value ≤0.001; ns means no significance.</p

Comparison pSTATs expression between SLE patient and healthy donor (HD) after cytokine stimulation.

<p>A representative experiment is shown. The MFI for the phosphorylation of STAT1 (5A), STAT3 (5B), and STAT5 (5C) in the T cells of SLE patient was compared with that of HD. The MFI for the phosphorylation of STAT1 (5D), STAT3 (5E), and STAT5 (5F) in the B cells of SLE patient was compared with that of HD. The MFI for the phosphorylation of STAT3 (5G) and STAT5 (5H) in the monocytes of SLE was compared with that of HD. The MFI for pSTAT3 (5I) in the T cells of SLE patient was compared with that of HD. MFI, mean fluorescence index; SLE, systemic lupus erythematosus; HD, healthy donor; IFNα, interferon α.</p

Comparison of pSTATs expression between SLE, RA patients and healthy donors (HDs).

<p>Each symbol represents an individual patient or HDs. (A) The MFI of pSTAT3 was elevated in the T cells of SLE, RA patients relative to that in HDs. (B) The MFI of pSTAT3 was elevated in the monocytes of SLE patients, but decreased in the monocytes of RA as compared with that in HDs. (C) The B cells of SLE patients, RA patients showed a higher MFI for pSTAT5 than that in HDs. (D) The T cells of SLE patients, RA patients showed a higher MFI for pSTAT5 than that in HDs. MFI, mean fluorescence index; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis;HDs, healthy donors.</p

Comparison of SLE patients in the two clusters.

<p>Values presented are means ± SD or numbers (%) of patients, depending on whether the data are continuous or dichotomous. ESR = erythrocyte sedimentation rate; Hgb = hemoglobin; dsDNA = double-stranded DNA; HCQ = hydroxychloroquine; NS = not significant.</p><p>†Mann–Whitney test was used, instead of a t test, because the data were not normally distributed.</p><p>¥Autoantibodies included anti-dsDNA, anti-Ro, anti-La, anti-U1 RNP, and anti-Sm antibodies, with a range of 0–5.</p><p>§All glucocorticoid doses were converted to equivalent daily doses of prednisone.</p

SLE samples were clustered by the expression of basal phosphorylated STAT3 and STAT5.

<p>Each grid represents the phosphorylation of a STAT protein in one cell type. The phosphorylation of each STAT protein was scaled relative to the minimum phosphorylation level among the 20 SLE samples.The 20 SLE patient samples were grouped according to their similarities using hierarchical clustering. Two groups of SLE patients were identified, based on similarities in the expression of basal phosphorylated STAT3 and STAT5 in specific cell types. HCQ use and the mean disease activity index (SLEDAI)-2K of each group are indicated at the bottom. SLE, systemic lupus erythematosus;HCQ, hydroxychloroquine.</p